Morbidity and mortality due to cardiovascular disease is substantially reduced by hormone replacement therapy in postmenopausal women. Evidence is mounting that demonstrates a beneficial effect of estrogen on the pathobiology of coronary artery disease, including improvement in endothelial vasomotor function. The underlying hypothesis is four-fold: (1) estrogen induces a favorable activation profile in endothelial cells (EC); (2) this includes augmentation of basal endothelial nitric oxide (NO) release; (3) non-genomic effects of estrogen receptors (ERs) are responsible for these events; and (4) kinase signaling cascades are involved. Based on our prior observations that human EC have functional ERs, a subset of which are plasma membrane-associated, and that l7beta-estradiol (E2) rapidly induces Ca2+-independent, tyrosine kinase-dependent endothelial nitric oxide synthase (eNOS) and MAP kinase activation (which are functionally linked), specific proposals now include (1) to determine the biochemical link between protein kinases, eNOS activity modulation and Ca2+-independence in estrogen-stimulated human umbilical vein EC (HUVEC) by assessing induced eNOS phosphorylation status, effects of MAP kinase and src inhibition on eNOS activity, and confirm E2-induced Hsp90-eNOS association (recently shown to enhance eNOS function); (2) to define which ER domains are critical in the rapid EC signaling response, measuring NO release from E2-stimulated cells transfected with ERalpha and beta constructs deleted in critical domains; and (3) to further delineate the structure and function of EC membrane ERs, using functional assays, immunolocalization in recombinant ER-transfected cells, biochemical definitions from purified plasma membranes, and yeast 2-hybrid screening for membrane ER-associated proteins. Information generated in these studies will both enhance our understanding of estrogen receptor and eNOS biology, and provide insight into their protective roles in the cardiovascular status of women.